The TIM gene family was identified using a congenic mouse model in which polymorphisms in TIM-1 and TIM-3 were associated with differences in Th1-Th2 differentiation and allergen-induced airway hyperreactivity (AHR) between BALB/c and congenic HBA mice. Our goal is to understand how the Tim genes regulate peripheral tolerance, adaptive immune responses, and allergy. We have shown that TIM-1 is an important costimulatory molecule for T cells, that TIM-1 and TIM-4 regulate T cell responses and the development of tolerance, and that TIM-1 and TIM-4 are receptors for phosphatidylserine (PtdSer), a key molecule for recognition and uptake of apoptotic cells. We have recently found that TIM-3, expressed on Th1 cells and APC, is also a receptor for PtdSer and that the allelic variants of TIM-3 associated with asthma differ in binding to PtdSer. These results suggest a new paradigm for TIM proteins as PtdSer receptors that by regulating the recognition, clearance, and response to apoptotic cells, can regulate T cell responses and the induction of peripheral tolerance. To understand how TIM protein recognition of PtdSer regulates immune responses, we propose to: Specific Aim 1: Determine the functional consequences of TIM-4 and TIM-3 on APC binding to phosphatidylserine on apoptotic cells. We will examine effects of apoptotic cell engulfment on DCs and macrophages including (a) anti-inflammatory mediator production: IL-10, TGF-B, IDO; (b) expression of co-inhibitory molecules; and (c) expression of co-stimulatory molecules. We will determine the effects of TIM-4- and TIM-3 mediated phagocytosis of apoptotic cell antigen on TReg and T helper subset development. Specific Aim 2. Determine the structure/function relationship of TIM allelic variants on recognition of phosphatidylserine, phagocytosis, and T cell activation. We will determine if cells expressing HBA and BALB/c alleles of TIM-1 and TIM-3 have different capacities to recognize PtdSer and phagocytose apoptotic cells. We will determine how binding of PtdSer on an apoptotic cell to TIM-1 and TIM-3 on a T cell regulates T cell activation and TReg development, and compare BALB/c and HBA T cells. Specific Aim 3: Investigate the in vivo role of TIM-1 and TIM-3 allelic variants in immune responses and regulation of peripheral tolerance. We will compare the roles of the TIM-3 pathway in clearance of apoptotic cells in vivo and in the presentation of apoptotic cell-associated antigen in BALB/c and HBA mice. We will determine the relative roles of TIM-1 and TIM-3 in regulating the development of AHR in BALB/c and HBA mice. These studies will greatly increase our understanding of the function of TIMs in the regulation of T cell responses and tolerance, and characterize a novel and extremely important asthma susceptibility gene family.